Acanthamoeba continuously releases lysosomal hydrolases into its growth medium. This release is specific for lysosomal hydrolases and is energy dependent. Hydrolases secreted by pinocytosing cells separate into two classes: one released at about 17% of the cellular content per hour and the other at about 4% per hour. We have suggested that hydrolase secretion is a consequence of membrane returning to the cell surface from digestive vacuoles via shuttle vesicles. When the vesicles fragment from the acidic digestive vacuoles, small amounts of the hydrolases are trapped in the vesicle lumen. Some of the hydrolases that have a higher affinity for membranes are bound to the vesicle membrane as well as being trapped in the lumen. When shuttle vesicles fuse with the plasma membrane the luminal contents are released and the vesicle membrane now exposed to the growth medium experiences a significant pH change. This change in pH decreases the affinity of hydrolase for membranes and causes the membrane-bound hydrolases to be unloaded. Hydrolases trapped in the shuttle vesicle lumen are released at low rates and form one of the classes of hydrolases. Hydrolases bound to the vesicle membranes as well as being trapped in the lumen are released at higher rates and form the second class of hydrolases. This model is supported by recent data. The pattern of differential hydrolases secretion, characteristic of pinocytosing amoebae, is lost when cells are incubated in growth media containing chloroquine or ammonium acetate, agents which raise the intravacuolar pH. Additionally, this pattern of hydrolase secretion is also lost by amoebae that have become saturated with yeast. This latter change in hydrolase secretion occurs concomitantly with an increase in vacuolar pH. All of these observations are consistent with our hypothesis that vacuolar pH is a factor regulating hydrolase secretion.